Journal: bioRxiv

Article Title: DEAD-box protein family member DDX28 is a negative regulator of HIF-2α and eIF4E2-directed hypoxic translation

doi: 10.1101/632331

Figure Lengend Snippet: (A) Western blot of total HIF-2α and DDX28 protein levels in normoxia (21% O 2 ) and hypoxia (1% O 2 ). GAPDH used as a loading control. (B) Western blot of DDX28 and HIF-2α (arrow, hypoxia-inducible lower band) normoxic and hypoxic protein levels in control (Ctrl) cells stably expressing a non-targeting shRNA or in cells stably expressing one of two shRNAs targeting DDX28 mRNA: Knockdown (KD) 1 and KD2. Actin used as a loading control. (C) GFP co-immunoprecipitation in hypoxic cells stably expressing FLAG-DDX28 and transfected with recombinant GFP-HIF-2α. Cells transfected with no DNA (−) or GFP alone were used as controls. (D) FLAG co-immunoprecipitation of recombinant FLAG-eIF4E2 from hypoxic cells co-transfected with HA-HIF-2α. Cells transfected with empty FLAG vector used as control. (E) FLAG co-immunoprecipitation in hypoxic cells stably expressing FLAG-DDX28 and transfected with recombinant HA-HIF-1α. Cells transfected with empty FLAG vector used as control. SE, short exposure; LE, long exposure. 25 μg of whole cell lysate was used as input. Experiments performed in U87MG glioblastoma.

Article Snippet: U87MG cells stably expressing C-terminal 3x FLAG tagged DDX28 were generated by transfecting cells with the OmicsLinkTM pEZ-M14 EX-A3144-M14 expression vector encoding the human DDX28 coding sequence (Genecopoeia).

Techniques: Western Blot, Control, Stable Transfection, Expressing, shRNA, Knockdown, Immunoprecipitation, Transfection, Recombinant, Plasmid Preparation

Journal: bioRxiv

Article Title: DEAD-box protein family member DDX28 is a negative regulator of HIF-2α and eIF4E2-directed hypoxic translation

doi: 10.1101/632331

Figure Lengend Snippet: Western blot and quantification of eIF4E2 capture with m 7 GTP-bound agarose beads in cells stably expressing one of two distinct shRNA sequences targeting DDX28 in 1% O 2 hypoxia (A-B) and 21% O 2 normoxia (C-D). 35 μg of whole cell lysate was used as the input. Ctrl, control cells stably expressing non-targeting shRNA; KD1 and KD2, knockdown cells stably expressing one of two distinct shRNA sequences targeting DDX28; LE, long exposure. Data (n ≥ 3), mean ± s.e.m normalized to input. * represents p < 0.05 using one sample t-test against hypothetical mean (μ = 1). Experiments performed in U87MG glioblastoma.

Article Snippet: U87MG cells stably expressing C-terminal 3x FLAG tagged DDX28 were generated by transfecting cells with the OmicsLinkTM pEZ-M14 EX-A3144-M14 expression vector encoding the human DDX28 coding sequence (Genecopoeia).

Techniques: Western Blot, Stable Transfection, Expressing, shRNA, Control, Knockdown

Journal: bioRxiv

Article Title: DEAD-box protein family member DDX28 is a negative regulator of HIF-2α and eIF4E2-directed hypoxic translation

doi: 10.1101/632331

Figure Lengend Snippet: Polysomal distribution of DDX28, eIF4E2 and eIF4E protein measured by western blot in control cells stably expressing non-targeting shRNA in 21% O 2 normoxia (A) and 1% O 2 hypoxia (C) and in Knockdown (KD) cells stably expressing an shRNA targeting DDX28 in normoxia (B) and hypoxia (D). Ribosomal protein L5 (rpL5) used as a marker of protein integrity in each fraction. The eIF4E (E) or eIF4E2 (F) protein associated with polysomes (fractions 4-9) as a percentage of total protein (fractions 1-9) was quantified by densitometry. Data (n = 3), mean ± s.e.m. * represents p < 0.05 using one-way ANOVA and Tukey’s HSD post-hoc test. Experiments performed in U87MG glioblastoma.

Article Snippet: U87MG cells stably expressing C-terminal 3x FLAG tagged DDX28 were generated by transfecting cells with the OmicsLinkTM pEZ-M14 EX-A3144-M14 expression vector encoding the human DDX28 coding sequence (Genecopoeia).

Techniques: Western Blot, Control, Stable Transfection, Expressing, shRNA, Knockdown, Marker

Journal: bioRxiv

Article Title: DEAD-box protein family member DDX28 is a negative regulator of HIF-2α and eIF4E2-directed hypoxic translation

doi: 10.1101/632331

Figure Lengend Snippet: ( A) Western blot of HIF-2α protein levels in cytoplasmic and nuclear fractions of control cells expressing a non-targeting shRNA or Knockdown (KD) cells stably expressing an shRNA targeting DDX28 in 1% O 2 hypoxia. Lamin a/c used as nuclear marker and α-tubulin as cytoplasmic marker. (B) The mRNA abundance of HIF-2α gene targets in hypoxia measured via qRT-PCR. Data (n ≥ 3), mean ± s.e.m. represented as log 2 (fold change) in DDX28 KD cells relative to control cells and normalized to endogenous control genes RPLP0 and RPL13A. * represents p < 0.05 using a one-sample t-test against hypothetical mean (μ = 0). CITED2, Cbp/P300-Interacting Transactivator 2; EPO, Erythropoietin; IGFBP3, Insulin Like Growth Factor Binding Protein 3; ITPR1, inositol 1,4,5-trisphosphate receptor type 1; LOXL2, Lysyl Oxidase Like 2; OCT4, octamer-binding transcription factor 4. Experiments performed in U87MG glioblastoma.

Article Snippet: U87MG cells stably expressing C-terminal 3x FLAG tagged DDX28 were generated by transfecting cells with the OmicsLinkTM pEZ-M14 EX-A3144-M14 expression vector encoding the human DDX28 coding sequence (Genecopoeia).

Techniques: Western Blot, Control, Expressing, shRNA, Knockdown, Stable Transfection, Marker, Quantitative RT-PCR, Binding Assay

Journal: bioRxiv

Article Title: DEAD-box protein family member DDX28 is a negative regulator of HIF-2α and eIF4E2-directed hypoxic translation

doi: 10.1101/632331

Figure Lengend Snippet: Viable cell counts were measured with crystal violet staining after 24 h, 48 h, and 72 h in 21% O 2 normoxia (A) and 1% O 2 hypoxia (B) for control cells expressing a non-targeting shRNA or Knockdown cells stably expressing one of two shRNAs targeting DDX28 (KD1 and KD2). All absolute cell count values were normalized to the number of cells present on day 0 for each individual independent experiment, representing the fold change in the number of cells at each time point relative to day 0. Proliferation was measured as % BrdU-positive control and DDX28 KD cells via immunofluorescence after 24 h in normoxia (C) or hypoxia (D). Data (n ≥ 3), mean ± s.e.m. * represents p < 0.05 using an unpaired two-sample t-test. Experiments performed in U87MG glioblastoma.

Article Snippet: U87MG cells stably expressing C-terminal 3x FLAG tagged DDX28 were generated by transfecting cells with the OmicsLinkTM pEZ-M14 EX-A3144-M14 expression vector encoding the human DDX28 coding sequence (Genecopoeia).

Techniques: Staining, Control, Expressing, shRNA, Knockdown, Stable Transfection, Cell Counting, Positive Control, Immunofluorescence

Journal: Neurotherapeutics

Article Title: Neuron-glioma synaptic transmission amplified by free 19S proteasome-mediated AMPAR deubiquitination promotes tumor progression

doi: 10.1016/j.neurot.2026.e00847

Figure Lengend Snippet: Free 19S Proteasomes Modulate Neuron-Glioma Synaptic Signaling. (A) Flow cytometric analysis of free 19S proteasomes (PSMD6 + PSMA7 - ) in EGFP + GL261 glioma cells under indicated conditions (n = 3). (B) Flow cytometric analysis of free 20S proteasomes (PSMA7 + PSMD6 - ) in EGFP + GL261 glioma cells under indicated conditions (n = 3). (C) Representative fluorescence microscopy images of CM-DiI-labeled GL261 cells (red) stained with membrane potential probe M09 (green) (n = 3). Scale bars: 100 μm. (D) Quantitative analysis of M09 MFI relative to control. Data normalized to monoculture group (set as 1.0) (n = 3). (E) Flow cytometric analysis of M09 signal in CM-DiI + GL261 cells under indicated conditions. (F) Percentage of M09-positive cells in CM-DiI + GL261 populations across treatment groups (n = 3). Data are means ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: The mouse glioma cell lines GL261 (YC–C045, RRID: CVCL_Y003) and GL261-EGFP (YC–C045-EGFP-P) were purchased from Ubigene (Shanghai, China).

Techniques: Fluorescence, Microscopy, Labeling, Staining, Membrane, Control

Journal: Neurotherapeutics

Article Title: Neuron-glioma synaptic transmission amplified by free 19S proteasome-mediated AMPAR deubiquitination promotes tumor progression

doi: 10.1016/j.neurot.2026.e00847

Figure Lengend Snippet: Free 19S Proteasomes Stabilize AMPAR through Deubiquitination. (A) Representative immunofluorescence images of 19S proteasomes (green, anti-PSMD6) and AMPAR subunits (red, anti-GluA1/GluA2) in glioma-infiltrated regions. Scale bars: 100 μm. (B) Quantification of GluA1 (left) and GluA2 (right) colocalization with PSMD6 in (A). (C) Correlation analysis of PSMD6 with GluA1 (left) and GluA2 (right) expression in GBM from GEPIA2 database. Spearman's correlation was used to assess the relationship between genes. (D, E) Flow cytometric analysis of surface GluA1 (D) and GluA2 (E) expression in EGFP + GL261 cells under indicated conditions (n = 3). (F, G) Flow cytometric analysis of surface GluA1 (F) and GluA2 (G) expression in EGFP + GL261 cells after bAP15 treatment (10 μM, 24h) (n = 3). Data are means ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: The mouse glioma cell lines GL261 (YC–C045, RRID: CVCL_Y003) and GL261-EGFP (YC–C045-EGFP-P) were purchased from Ubigene (Shanghai, China).

Techniques: Immunofluorescence, Expressing

Journal: Neurotherapeutics

Article Title: Neuron-glioma synaptic transmission amplified by free 19S proteasome-mediated AMPAR deubiquitination promotes tumor progression

doi: 10.1016/j.neurot.2026.e00847

Figure Lengend Snippet: The free 19S-AMPAR Axis Promotes Glioma Proliferation and Invasion. (A) Representative immunofluorescence images of Ki67 in EGFP + GL261 cells under indicated conditions (bAP15 = 10 μM; CNQX = 10 μM). Scale bars: 100 μm. (B) Quantitative analysis of Ki67 mean fluorescence intensity relative to control. Data normalized to monoculture group (set as 1.0) (n = 3). (C) Proliferation rate of EGFP + GL261 cells measured by fluorescence intensity over 72 h (n = 5). (D) Quantification of invaded cells in Transwell assays. Data expressed as cell number per field (n = 3). (E) Representative images of invaded EGFP + GL261 cells in Transwell assays under indicated conditions. Scale bars: 200 μm. Data are means ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: The mouse glioma cell lines GL261 (YC–C045, RRID: CVCL_Y003) and GL261-EGFP (YC–C045-EGFP-P) were purchased from Ubigene (Shanghai, China).

Techniques: Immunofluorescence, Fluorescence, Control

Journal: Neurotherapeutics

Article Title: Neuron-glioma synaptic transmission amplified by free 19S proteasome-mediated AMPAR deubiquitination promotes tumor progression

doi: 10.1016/j.neurot.2026.e00847

Figure Lengend Snippet: Targeting the Free 19S-AMPAR Axis Sensitizes Gliomas to Radiotherapy. (A) Flow cytometric analysis of total 19S proteasomes (PSMD6 + ) expression in EGFP + GL261 glioma cells from the NGS model treated with or without 6 Gy radiotherapy (n = 3). (B) Flow cytometric analysis of free 19S proteasomes (PSMD6 + PSMA7 - ) expression in EGFP + GL261 glioma cells from the NGS model treated with or without 6 Gy radiotherapy (n = 3). (C, D) Flow cytometric analysis of surface GluA1 (C) and GluA2 (D) expression in EGFP + GL261 glioma cells from the NGS model under indicated conditions (n = 3). (E) Representative immunofluorescence images of Ki67 in EGFP + GL261 cells from the NGS model. Scale bars: 100 μm. (F) Quantitative analysis of Ki67 MFI relative to control. Data normalized to monoculture group (set as 1.0) (n = 3). (G) Proliferation kinetics of EGFP + GL261 cells measured by fluorescence intensity over 72 h (n = 5). (H) Representative images of invaded EGFP + GL261 cells in Transwell assays. Scale bars: 200 μm. (I) Quantification of invaded cells in Transwell assays (n = 3). Data expressed as cell number per field. Data are means ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: The mouse glioma cell lines GL261 (YC–C045, RRID: CVCL_Y003) and GL261-EGFP (YC–C045-EGFP-P) were purchased from Ubigene (Shanghai, China).

Techniques: Expressing, Immunofluorescence, Control, Fluorescence

Journal: Toxicological sciences : an official journal of the Society of Toxicology

Article Title: Chaperone proteins involved in troglitazone-induced toxicity in human hepatoma cell lines.

doi: 10.1093/toxsci/kfi022

Figure Lengend Snippet: FIG. 1. TZDs cytotoxicity in HepG2 cells. (A) HepG2 cells were incubated with 0.1% DMSO (control) or the indicated concentration of TRO or RSG for 48 h. (B) HepG2 cells were incubated with 0.1% DMSO (control) or 50 mM TZDs for the indicated time. Cell viabilities were assessed by crystal violet assay. (C) HepG2 cells were incubated with 0.1% DMSO (control) or the indicated concentration of TRO or RSG for 48 h. Cell viabilities were measured by ATP- based luminescent assay. The results represent the mean 6 SD of at least three independent experiments. *p 5 0.05, **p 5 0.01 compared with the control.

Article Snippet: Human hepatoma cell lines HepG2 and HLE were obtained from the Riken Gene Bank (Tsukuba, Japan, http://www.rtc.riken.go.jp) and the Japanese Collection of Research Biosources (Tokyo, Japan, http://cellbank.nihs.go.jp/cellbank_e.html), respectively.

Techniques: Incubation, Control, Concentration Assay, Crystal Violet Assay, Luminescence Assay

Journal: Toxicological sciences : an official journal of the Society of Toxicology

Article Title: Chaperone proteins involved in troglitazone-induced toxicity in human hepatoma cell lines.

doi: 10.1093/toxsci/kfi022

Figure Lengend Snippet: FIG. 2. Protein expression profiles of 2-D gels from CBB staining. HepG2 cells were incubated with 0.1% DMSO (control) or the indicated concentration of TRO or RSG for 48 h. The cell lysates were separated by 2-DE and stained with CBB. The oval frames mark the proteins of interest, which were increased in a concentration-dependent manner by TRO treatment.

Article Snippet: Human hepatoma cell lines HepG2 and HLE were obtained from the Riken Gene Bank (Tsukuba, Japan, http://www.rtc.riken.go.jp) and the Japanese Collection of Research Biosources (Tokyo, Japan, http://cellbank.nihs.go.jp/cellbank_e.html), respectively.

Techniques: Expressing, Staining, Incubation, Control, Concentration Assay

Journal: Toxicological sciences : an official journal of the Society of Toxicology

Article Title: Chaperone proteins involved in troglitazone-induced toxicity in human hepatoma cell lines.

doi: 10.1093/toxsci/kfi022

Figure Lengend Snippet: FIG. 4. Western blot analyses. HepG2 cells were incubated with 0.1% DMSO (control) or the indicated concentrations of TRO or RSG for 48 h. The cell lysates were subjected to Western blot analysis with mouse anti-KDEL (A), rabbit anti-ERp72 (B), and rabbit anti-PDI (C) antibodies for BiP, PDIrp, and PDI proteins, respectively. Two additional studies yielded equivalent results. The expressions of BiP, PDIrp, and PDI proteins were measured using a densitometer. The data represent the mean 6 SD of three independent experiments. **p 5 0.01 compared with the control.

Article Snippet: Human hepatoma cell lines HepG2 and HLE were obtained from the Riken Gene Bank (Tsukuba, Japan, http://www.rtc.riken.go.jp) and the Japanese Collection of Research Biosources (Tokyo, Japan, http://cellbank.nihs.go.jp/cellbank_e.html), respectively.

Techniques: Western Blot, Incubation, Control

Journal: Toxicological sciences : an official journal of the Society of Toxicology

Article Title: Chaperone proteins involved in troglitazone-induced toxicity in human hepatoma cell lines.

doi: 10.1093/toxsci/kfi022

Figure Lengend Snippet: FIG. 5. Changes of BiP and PDIrp mRNA expression by TZDs in HepG2 cells. HepG2 cells were incubated with 0.1% DMSO (control) or the indicated concentrations of TRO or RSG for 48 h. The cDNA were prepared for real-time PCR. Sense and antisense primers of BiP (50-TGCTTGATGTATGTCCCCTTA-30

Article Snippet: Human hepatoma cell lines HepG2 and HLE were obtained from the Riken Gene Bank (Tsukuba, Japan, http://www.rtc.riken.go.jp) and the Japanese Collection of Research Biosources (Tokyo, Japan, http://cellbank.nihs.go.jp/cellbank_e.html), respectively.

Techniques: Expressing, Incubation, Control, Real-time Polymerase Chain Reaction